This invention relates to a novel method for inhibiting microvascular thrombosis and, more particularly, to a method for reducing the thrombogenicity of microvascular anastomoses during microvascular reconstruction by the topical administration of a blood coagulation inhibitor known as lipoprotein-associated coagulation inhibitor (LACI) and alternatively as tissue factor pathway inhibitor (TFPI).
Thrombosis of microvascular anastomoses, particularly in cases of extremity trauma in free flap reconstructions, is a significant problem for the reconstructive surgeon. A recent survey by Salemark, Microsurgery 12, 308-311 (1991), revealed that many centers routinely make use of systemic anticoagulation for replants and free flap transfers. However, the risk for generalized hemorrhage [Leung, Hand 12, 25-32 (1980); Hirsh, Semin. Thromb. Hemostas. 12, 21-32 (1980)], with compounding risks from blood transfusion products, leaves open the question of benefit from massive systemic circulatory alteration merely to preserve flow in a small vessel supplying blood to non-vital tissue.
The concept of site-specific action by an antithrombotic agent, administered through simple topical application was proposed by Cooley and Gould, Microsurgery 12, 281-287 (1991). Since those vessels which are prone to thrombosis are exposed during the reconstructive effort, with ready access to the lumenal surface during the anastomotic repair, an agent could be applied through the course of normal vessel irrigation, potentially achieving a highly localized effect through surface binding to the thrombogenic site(s). In fact, Cooley et al. have described one possible agent, a peptide based on the platelet-binding and fibrin-polymerizing region of fibrinogen, and have shown its ability to reduce the occurrence of thrombotic occlusion in a rat model [6th Ann. Meeting, Amer. Soc. Reconstructive Microsurgery, Toronto, Canada. Sep. 21-23, 1990].
Thrombosis caused by vascular injury is at least partially if not predominantly initiated through the tissue-factor-mediated pathway of coagulation.
Plasma contains a multivalent Kunitz-type inhibitor of coagulation, referred to herein as tissue factor pathway inhibitor (TFPI). This name has been accepted by the International Society on Thrombosis and Hemostasis, Jun. 30, 1991, Amsterdam. TFPI was first purified from a human hepatoma cell, Hep G2, as described by Broze and Miletich, Proc. Natl. Acad. Sci. USA 84, 1886-1890 (1987), and subsequently from human plasma as reported by Novotny et al., J. Biol. Chem. 264, 18832-18837 (1989); Chang liver and SK hepatoma cells as disclosed by Wun et al., J. Biol. Chem. 265, 16096-16101 (1990). TFPI cDNA have been isolated from placental and endothelial cDNA libraries as described by Wun et al., J. Biol. Chem. 263, 6001-6004 (1988); Girard et al., Thromb. Res. 55, 37-50 (1989). The primary amino acid sequence of TFPI, deduced from the cDNA sequence, shows that TFPI contains a highly negatively charged amino-terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus. The first Kunitz-domain of TFPI is needed for the inhibition of the factor VII.sub.a /tissue factor complex and the second Kunitz-domain of TFPI is responsible for the inhibition of factor X.sub.a according to Girard et al., Nature 328, 518-520 (1989), while the function of the third Kunitz-domain remains unknown. See also copending application Ser. No. 07/301,779, filed Jan. 26, 1989, now U.S. Pat. No. 5,106,833. TFPI is believed to function in vivo to limit the initiation of coagulation by forming an inert, quaternary factor X.sub.a : TFPI: factor VII.sub.a : tissue factor complex. Further background information on TFPI can be had by reference to the recent reviews by Rapaport, Blood 73, 359-365 (1989); Broze et al., Biochemistry 29, 7539-7546 (1990).
Recombinant TFPI has been expressed as a glycosylated protein using mammalian cell hosts including mouse C127 cells as disclosed by Day et al., Blood 76, 1538-1545 (1990), baby hamster kidney cells as reported by Pedersen et al., J. Biol. Chem. 265, 16786-16793 (1990), Chinese hamster ovary cells and human SK hepatoma cells. The C127 TFPI has been used in animal studies and shown to be effective in the inhibition of tissue factor-induced intravascular coagulation in rabbits according to Day et al., supra, and in the prevention of arterial reocclusion after thrombolysis in dogs as described by Haskel et al., Circulation 84, 821-827 (1991).
Recombinant TFPI also has been expressed as a non-glycosylated protein using E. coli host cells and obtaining a highly active TFPI by in vitro folding of the protein as described in co-pending application of Judy A. Diaz-Collier, Mark E. Gustafson and Tze-Chein Wun, on "Method of Producing Tissue Factor Pathway Inhibitor", Ser. No. 07/844,297, filed Mar. 22, 1992, now U.S. Pat. No. 5,112,091, the disclosure of which is incorporated by reference herein.
The cloning of the TFPI cDNA which encodes the 276 amino acid residue protein of TFPI is further described in Wun et al., U.S. Pat. No. 4,966,852, the disclosure of which is incorporated by reference herein.